# US 8,541,172 B2 · Method for sequencing a polynucelotide template

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- Number: US 8,541,172 B2
- Document ID: US8541172B2
- Status: Granted
- Kind: B2
- Application number: 13/445,072
- Filed: 2012-04-12
- Published: 2012-10-11
- Granted: 2013-09-24
- Earliest claimed: 2009-09-15
- Assignees: Illumina Cambridge Limited
- Applicants: Alan D. Kersey; Jonathan Mark Boutell
- Inventors: Alan D. Kersey; Jonathan Mark Boutell
- Web page: https://patentgrep.com/patent/US8541172B2

## CPC

- C12Q 1/6874: involving nucleic acid arrays, e.g. sequencing by hybridisation
- C12Q 1/6806: Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay (C12Q1/6804 takes precedence)
- C12Q: MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS (immunoassay G01N33/53); COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES

## Abstract

Improved compositions, methods, apparatus, and kits for high-throughput nucleic acid amplification, detection and sequencing are disclosed. A nucleic acid cluster having an identifiable center is produced by generating on a solid support an immobilized nucleic acid complement from a template, one of which comprises a detectable label; and amplifying the complement and the template to obtain a nucleic acid cluster on the support, the cluster having a substantially central location marked by the detectable label and a surrounding region comprising immobilized copies. Also disclosed are nucleotide sequence determination in nucleic acid clusters so produced, center position annotation in the clusters, assignment of sequence information to overlapping clusters, and related compositions and methods.

## Claims

### Claim 1

A method for sequencing nucleic acids, comprising
(a) attaching a plurality of different nucleic acids to a surface;
(b) amplifying the different nucleic acids on the surface to form a plurality of clusters on the surface, wherein clusters in the plurality of clusters abut or overlap with each other on the surface, wherein each of the clusters comprises copies of a single nucleic acid from the plurality of different nucleic acids; and
(c) determining the sequences of the amplified copies in the clusters, wherein the sequences are determined by a method comprising:
(i) obtaining images of overlapping signals emanating from the clusters that abut or overlap with each other on the surface, and
(ii) processing the images to assign nucleic acid sequence information to a single cluster, wherein one or more nucleic acids in a cluster are distinguished from surrounding nucleic acids in the same cluster.

### Claim 2 (depends on 1)

The method of claim 1, wherein the surface is contained in a flow cell.

### Claim 3 (depends on 1)

The method of claim 1, wherein the amplifying in (b) is isothermal amplification.

### Claim 4 (depends on 1)

The method of claim 1, wherein the diameter of each of the clusters is between 0.2 μm to 6 μm.

### Claim 5 (depends on 1)

The method of claim 1, wherein the density of the plurality of clusters is in the range from 100,000 clusters per mm2 to 10,000,000 clusters per mm2.

### Claim 6 (depends on 1)

The method of claim 1, wherein the density of the plurality of clusters is in the range from 1,000,000 clusters per mm2 to 10,000,000 clusters per mm2.

### Claim 7 (depends on 1)

The method of claim 1, wherein the clusters comprise a circular shape.

### Claim 8 (depends on 1)

The method of claim 1, wherein the clusters comprise a shape other than a circular shape.

### Claim 9 (depends on 1)

The method of claim 1, wherein the amplifying uses only one species of oligonucleotide primers immobilized on the surface.

### Claim 10 (depends on 1)

The method of claim 1, wherein the amplifying uses first and second different species of oligonucleotide primers immobilized on the surface.

### Claim 11 (depends on 1)

The method of claim 1, wherein the image detects 100,000,000 or more clusters simultaneously.

### Claim 12 (depends on 1)

The method of claim 1, wherein the one or more nucleic acids in the cluster that are distinguished from the surrounding nucleic acids in the same cluster comprise the identifiable center of the cluster.

### Claim 13 (depends on 1)

The method of claim 1, wherein the determining of the sequences comprises determining a reference position in each of the clusters, wherein the reference position distinguishes the signals detected from the clusters that abut or overlap with each other.

### Claim 14 (depends on 13)

The method of claim 13, wherein the reference position of each of the clusters is surrounded by a region of the respective cluster.

### Claim 15 (depends on 13)

The method of claim 13, wherein the reference position of each of the clusters comprises the identifiable center of the cluster.

### Claim 16 (depends on 1)

The method of claim 1, wherein the determining of the sequences comprises sequencing by synthesis.

### Claim 17 (depends on 1)

The method of claim 1, wherein the determining of the sequences comprises sequencing by ligation.

### Claim 18 (depends on 1)

The method of claim 1, wherein the determining of the sequences comprises repeated cycles of the obtaining of the images and the processing of the images.

### Claim 19 (depends on 1)

The method of claim 1, wherein the signals comprise fluorescent signals.

### Claim 20 (depends on 1)

The method of claim 1, wherein each of the clusters comprises at least 500 copies of a single nucleic acid from the plurality of different nucleic acids.

### Claim 21 (depends on 1)

The method of claim 1, wherein a plurality of oligonucleotide primers is immobilized on the surface.

### Claim 22 (depends on 21)

The method of claim 21, wherein the attaching comprises hybridizing the different nucleic acids to the oligonucleotide primers that are immobilized on the surface.

### Claim 23 (depends on 22)

The method of claim 22, wherein the plurality of different nucleic acids comprise universal sequences that hybridize to the oligonucleotide primers that are immobilized on the surface.

### Claim 24 (depends on 23)

The method of claim 23, wherein the universal sequences are present at the 5′ ends of the different nucleic acids.

### Claim 25 (depends on 23)

The method of claim 23, wherein the universal sequences are present at the 3′ ends of the different nucleic acids.

### Claim 26

An automated system comprising a processor and a non-transitory computer readable medium comprising instructions for
(a) attaching a plurality of different nucleic acids to a surface;
(b) amplifying the different nucleic acids on the surface to form a plurality of clusters on the surface, wherein clusters in the plurality of clusters abut or overlap with each other on the surface, wherein each of the clusters comprises copies of a single nucleic acid from the plurality of different nucleic acids; and
(c) determining the sequences of the amplified copies in the clusters, wherein the sequences are determined by a method comprising
(i) obtaining images of overlapping signals emanating from the clusters that abut or overlap with each other on the surface, and
(ii) processing the image to assign nucleic acid sequence information to a single cluster, wherein one or more nucleic acids in a cluster are distinguished from surrounding nucleic acids in the same cluster.